Team Publications UMR3244 – Dynamics of Genetic Information

Year of publication 2021

Ana Martins Figueiredo, Pedro Barbacena, Ana Russo, Silvia Vaccaro, Daniela Ramalho, Andreia Pena, Aida Pires Lima, Rita Rua Ferreira, Marta Alves Fidalgo, Fatima El-Marjou, Yulia Carvalho, Francisca Ferreira Vasconcelos, Ana-Maria Lennon-Duménil, Danijela Matic Vignjevic, Claudio Areias Franco (2021 Apr 27) Endothelial cell invasion is controlled by dactylopodia. Proceedings of the National Academy of Sciences of the United States of America : DOI : e2023829118

Summary

Sprouting angiogenesis is fundamental for development and contributes to cancer, diabetic retinopathy, and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on how tip cells invade into tissues. Here, we show that endothelial tip cells use dactylopodia as the main cellular protrusion for invasion into nonvascular extracellular matrix. We show that dactylopodia and filopodia protrusions are balanced by myosin IIA (NMIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of NMIIA promotes excessive dactylopodia formation in detriment of filopodia. Conversely, endothelial cell- autonomous ablation of Arp2/3 prevents dactylopodia development and leads to excessive filopodia formation. We further show that NMIIA inhibits Rac1-dependent activation of Arp2/3 by regulating the maturation state of focal adhesions. Our discoveries establish a comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way toward strategies to block invasive tip cells during sprouting angiogenesis.

Shanna L Bowman, Linh Le, Yueyao Zhu, Dawn C Harper, Anand Sitaram, Alexander C Theos, Elena V Sviderskaya, Dorothy C Bennett, Graça Raposo-Benedetti, David J Owen, Megan K Dennis, Michael S Marks (2021 Apr 22) A BLOC-1-AP-3 super-complex sorts a cis-SNARE complex into endosome-derived tubular transport carriers. The Journal of cell biology : DOI : e202005173

Summary

Membrane transport carriers fuse with target membranes through engagement of cognate vSNAREs and tSNAREs on each membrane. How vSNAREs are sorted into transport carriers is incompletely understood. Here we show that VAMP7, the vSNARE for fusing endosome- derived tubular transport carriers with maturing melanosomes in melanocytes, is sorted into transport carriers in complex with the tSNARE component STX13. Sorting requires either recognition of VAMP7 by the AP-3δ subunit of AP-3 or of STX13 by the pallidin subunit of BLOC-1, but not both. Consequently, melanocytes expressing both AP-3δ and pallidin variants that cannot bind their respective SNARE proteins are hypopigmented and fail to sort BLOC-1-dependent cargo, STX13, or VAMP7 into transport carriers. However, SNARE binding

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does not influence BLOC-1 function in generating tubular transport carriers. These data reveal a novel mechanism of vSNARE sorting by recognition of redundant sorting determinants on a SNARE complex by an AP-3-BLOC-1 super-complex.

Abegão L.M., Santos F.A., Piguel S., Rodrigues J.J., Mendonça C.R., De Boni L. (2021 Apr 15) The ability of 2,5-disubstituted oxazole dyes derivatives to generate two-photon upconversion photoluminescence and its brightness evaluation Journal of Photochemistry and Photobiology A: Chemistry : 411 : 113214 : DOI : 10.1016/j.jphotochem.2021.113214

Summary

The brightness study of emissive compounds is one of the fundamental spectroscopic characterizations. In this work, we assessed the brightness values of eight 2,5-disubstituted oxazole dyes derivatives by combining linear and nonlinear spectroscopic parameters. The range of the brightness values obtained is from 0.26 GM to 15.15 GM. The highest value belongs to compound 14 m, which, compared to previously investigated compounds of similar π-conjugation length, is at least two times higher. Brightness values were determined in the spectral region between 700 nm–720 nm, revealing this class of dyes’ potential to be used as photoluminescence bioprobes excited by two-photons.

Patrick T Rudak, Joshua Choi, Katie M Parkins, Kelly L Summers, Dwayne N Jackson, Paula J Foster, Anton I Skaro, Ken Leslie, Vivian C McAlister, Vijay K Kuchroo, Wataru Inoue, Olivier Lantz, S M Mansour Haeryfar (2021 Apr 14)

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 2 Team Publications UMR3244 – Dynamics of Genetic Information

Chronic stress physically spares but functionally impairs innate-like invariant T cells. Cell reports : 108979 : DOI : S2211-1247(21)00293-X

Summary

The deleterious effects of psychological stress on mainstream T lymphocytes are well documented. However, how stress impacts innate-like T cells is unclear. We report that long- term stress surprisingly abrogates both T helper 1 (T1)- and T2-type responses orchestrated by invariant natural killer T (iNKT) cells. This is not due to iNKT cell death because these cells are unusually refractory to stress-inflicted apoptosis. Activated iNKT cells in stressed mice exhibit a “split” inflammatory signature and trigger sudden serum interleukin-10 (IL-10), IL-23, and IL-27 spikes. iNKT cell dysregulation is mediated by cell-autonomous glucocorticoid receptor signaling and corrected upon habituation to predictable stressors. Importantly, under stress, iNKT cells fail to potentiate cytotoxicity against lymphoma or to reduce the burden of metastatic melanoma. Finally, stress physically spares mouse mucosa- associated invariant T (MAIT) cells but hinders their T1-/T2-type responses. The above findings are corroborated in human peripheral blood and hepatic iNKT/MAIT cell cultures. Our work uncovers a mechanism of stress-induced immunosuppression.

Szuba Agata, Bano Fouzia, Castro Linares Gerard , Iv Francois, Mavrakis Manos*, Richter Ralf P*, Bertin Aurélie*, Koenderink Gijsje H* (2021 Apr 13) Membrane binding controls ordered self-assembly of animal septins eLifeeLife : eLife 2021;10:e63349 : DOI : 10.7554/eLife.63349

Summary

Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here we show by cell-free reconstitution that binding to flat lipid membranes requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septin hexamers form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12 to 18 nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4 nm thin monolayers, indicating that stacking requires the C- terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.

Ophélie Lautier, Arianna Penzo, Jérôme O Rouvière, Guillaume Chevreux, Louis Collet, Isabelle Loïodice, Angela Taddei, Frédéric Devaux, Martine A Collart, Benoit Palancade (2021 Apr 10) Co-translational assembly and localized translation of nucleoporins in nuclear

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pore complex biogenesis. Molecular cell : DOI : S1097-2765(21)00225-2

Summary

mRNA translation is coupled to multiprotein complex assembly in the cytoplasm or to protein delivery into intracellular compartments. Here, by combining systematic RNA immunoprecipitation and single-molecule RNA imaging in yeast, we have provided a complete depiction of the co-translational events involved in the biogenesis of a large multiprotein assembly, the nuclear pore complex (NPC). We report that binary interactions between NPC subunits can be established during translation, in the cytoplasm. Strikingly, the nucleoporins Nup1/Nup2, together with a number of nuclear proteins, are instead translated at nuclear pores, through a mechanism involving interactions between their nascent N- termini and nuclear transport receptors. Uncoupling this co-translational recruitment further triggers the formation of cytoplasmic foci of unassembled polypeptides. Altogether, our data reveal that distinct, spatially segregated modes of co-translational interactions foster the ordered assembly of NPC subunits and that localized translation can ensure the proper delivery of proteins to the pore and the nucleus.

Marion Blin, Laurent Lacroix, Nataliya Petryk, Yan Jaszczyszyn, Chun-Long Chen, Olivier Hyrien, Benoît Le Tallec (2021 Apr 9) DNA molecular combing-based replication fork directionality profiling. Nucleic acids research : DOI : gkab219

Summary

The replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas inefficient origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing. Importantly, we demonstrate that inefficient origins do not appear as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.

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Tsai Feng-Ching, Simunovic Mijo, Sorre Benoit , Bertin Aurélie, Manzi John, Callan-Jones Andrew, Bassereau Patricia (2021 Apr 6) Comparing physical mechanisms for membrane curvature-driven sorting of BAR- domain proteins Soft Matter : DOI : 10.1039/D0SM01573C

Summary

Protein enrichment at specific membrane locations in cells is crucial for many cellular functions. It is well-recognized that the ability of some proteins to sense membrane curvature contributes partly to their enrichment in highly curved cellular membranes. In the past, different theoretical models have been developed to reveal the physical mechanisms underlying curvature-driven protein sorting. This review aims to provide a detailed discussion of the two continuous models that are based on the Helfrich elasticity energy, (1) the spontaneous curvature model and (2) the curvature mismatch model. These two models are commonly applied to describe experimental observations of protein sorting. We discuss how they can be used to explain the curvature-induced sorting data of two BAR proteins, amphiphysin and centaurin. We further discuss how membrane rigidity, and consequently the membrane curvature generated by BAR proteins, could influence protein organization on the curved membranes. Finally, we address future directions in extending these models to describe some cellular phenomena involving protein sorting.

Dipti Vinayak Vernekar, Giordano Reginato, Céline Adam, Lepakshi Ranjha, Florent Dingli, Marie- Claude Marsolier, Damarys Loew, Raphaël Guérois, Bertrand Llorente, Petr Cejka, Valérie Borde (2021 Apr 6) The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length. Nucleic acids research : DOI : gkab232

Summary

Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLβ heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLβ recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLβ ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3- MutLβ may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1’s activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLβ

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ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.

Heltberg Mathias, Miné-Hattab Judith, Taddei Angela , Walczak Aleksandra M. , Mora Thierry (2021 Apr 2) Physical observables to determine the nature of membrane-less cellular sub- compartments preprint. : DOI : 10.1101/2021.04.01.438041

Summary

Abstract

The spatial organization of complex biochemical reactions is essential for the regulation of cellular processes. Membrane-less structures called foci containing high concentrations of specific proteins have been reported in a variety of contexts, but the mechanism of their formation is not fully understood. Several competing mechanisms exist that are difficult to distinguish empirically, including liquid-liquid phase separation, and the trapping of molecules by multiple binding sites. Here we propose a theoretical framework and outline observables to differentiate between these scenarios from single molecule tracking experiments. In the binding site model, we derive relations between the distribution of proteins, their diffusion properties, and their radial displacement. We predict that protein search times can be reduced for targets inside a liquid droplet, but not in an aggregate of slowly moving binding sites. These results are applicable to future experiments and suggest different biological roles for liquid droplet and binding site foci.

Yu Luo, Anton Granzhan, Daniela Verga, Jean-Louis Mergny (2021 Apr 1) FRET-MC: A fluorescence melting competition assay for studying G4 structures in vitro Biopolymers : 112 : e23415 : DOI : 10.1002/bip.23415

Summary

G-quadruplexes (G4) play crucial roles in biology, analytical chemistry and nanotechnology. The stability of G4 structures is impacted by the number of G-quartets, the length and positions of loops, flanking motifs, as well as additional structural elements such as bulges, capping base pairs, or triads. Algorithms such as G4Hunter or Quadparser may predict if a given sequence is G4-prone by calculating a quadruplex propensity score; however, experimental validation is still required. We previously demonstrated that this validation is not always straightforward, and that a combination of techniques is often required to unambiguously establish whether a sequence forms a G-quadruplex or not. In this article, we adapted the well-known FRET-melting assay to characterize G4 in batch, where the sequence

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to be tested is added, as an unlabeled competitor, to a system composed of a dual-labeled probe (F21T) and a specific quadruplex ligand. PhenDC3 was preferred over TMPyP4 because of its better selectivity for G-quadruplexes. In this so-called FRET-MC (melting competition) assay, G4-forming competitors lead to a marked decrease of the ligand-induced stabilization

effect (∆Tm), while non-specific competitors (e.g., single- or double-stranded sequences) have little effect. Sixty-five known sequences with different typical secondary structures were used to validate the assay, which was subsequently employed to assess eight novel sequences that were not previously characterized.

Zackie Aktary, Alejandro Conde-Perez, Florian Rambow, Mathilde Di Marco, François Amblard, Ilse Hurbain, Graça Raposo, Cédric Delevoye, Sylvie Coscoy, Lionel Larue (2021 Mar 27) A role for Dynlt3 in melanosome movement, distribution, acidity and transfer. Communications biology : 423 : DOI : 10.1038/s42003-021-01917-5

Summary

Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, acidity and transfer. In Mus musculus melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional motion, leading to their peripheral location in the cell. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is

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dependent on β-catenin activity, revealing a function of the Wnt/β-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, positioning and acidity of melanosomes required for their transfer.

Emeline Bonsergent, Eleonora Grisard, Julian Buchrieser, Olivier Schwartz, Clotilde Théry, Grégory Lavieu (2021 Mar 26) Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells. Nature communications : 1864 : DOI : 10.1038/s41467-021-22126-y

Summary

Extracellular vesicles (EVs), including exosomes, are thought to mediate intercellular communication through the transfer of cargoes from donor to acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. Here, we developed a cell-based assay in which EVs containing luciferase- or fluorescent-protein tagged cytosolic cargoes are loaded on unlabeled acceptor cells. Results from dose-responses, kinetics, and temperature-block experiments suggest that EV uptake is a low yield process (~1% spontaneous rate at 1 h). Further characterization of this limited EV uptake, through fractionation of membranes and cytosol, revealed cytosolic release (~30% of the uptaken EVs) in acceptor cells. This release is inhibited by bafilomycin A1 and overexpression of IFITM proteins, which prevent virus entry and fusion. Our results show that EV content release requires endosomal acidification and suggest the involvement of membrane fusion.

Alexandra Zak, Sara Violeta Merino-Cortés, Anaïs Sadoun, Farah Mustapha, Avin Babataheri, Stéphanie Dogniaux, Sophie Dupré-Crochet, Elodie Hudik, Hai-Tao He, Abdul I Barakat, Yolanda R Carrasco, Yannick Hamon, Pierre-Henri Puech, Claire Hivroz, Oliver Nüsse, Julien Husson (2021 Mar 17) Rapid viscoelastic changes are a hallmark of early leukocyte activation. Biophysical journal : 1692-1704 : DOI : S0006-3495(21)00200-9

Summary

To accomplish their critical task of removing infected cells and fighting pathogens, leukocytes activate by forming specialized interfaces with other cells. The physics of this key immunological process are poorly understood, but it is important to understand them because leukocytes have been shown to react to their mechanical environment. Using an innovative micropipette rheometer, we show in three different types of leukocytes that, when stimulated by microbeads mimicking target cells, leukocytes become up to 10 times stiffer and more viscous. These mechanical changes start within seconds after contact and evolve rapidly over minutes. Remarkably, leukocyte elastic and viscous properties evolve in parallel, preserving a well-defined ratio that constitutes a mechanical signature specific to each cell type. Our results indicate that simultaneously tracking both elastic and viscous

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properties during an active cell process provides a new, to our knowledge, way to investigate cell mechanical processes. Our findings also suggest that dynamic immunomechanical measurements can help discriminate between leukocyte subtypes during activation.

Elisa Le Boiteux, Franck Court, Pierre-Olivier Guichet, Catherine Vaurs-Barrière, Isabelle Vaillant, Emmanuel Chautard, Pierre Verrelle, Bruno M Costa, Lucie Karayan-Tapon, Anne Fogli, Philippe Arnaud (2021 Mar 15) Widespread overexpression from the four DNA hypermethylated HOX clusters in aggressive (IDHwt) glioma is associated with H3K27me3 depletion and alternative promoter usage. Molecular oncology : Accepted article : DOI : 10.1002/1878-0261.12944

Summary

In human, the 39 coding HOX genes and 18 referenced non-coding antisense transcripts are arranged in four genomic clusters named HOXA, B, C, and D. This highly conserved family belongs to the homeobox class of genes that encode transcription factors required for normal development. Therefore, HOX gene deregulation might contribute to the development of many cancer types. Here, we study HOX gene deregulation in adult glioma, a common type of primary brain tumor. We performed extensive molecular analysis of tumor samples, classified according to their isocitrate dehydrogenase (IDH1) gene mutation status, and of glioma stem cells. We found widespread expression of sense and antisense HOX transcripts only in aggressive (IDHwt) glioma samples, although the four HOX clusters displayed DNA hypermethylation. Integrative analysis of expression-, DNA methylation- and histone modification signatures along the clusters revealed that HOX gene upregulation relies on canonical and alternative bivalent CpG island promoters that escape hypermethylation. H3K27me3 loss at these promoters emerges as the main cause of widespread HOX gene upregulation in IDHwt glioma cell lines and tumors. Our study provides the first comprehensive description of the epigenetic changes at HOX clusters and their contribution to the transcriptional changes observed in adult glioma. It also identified putative “master” HOX proteins that might contribute to the tumorigenic potential of glioma stem cells.

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